Platelets play a central role in both hemostasis and thrombosis, and contribute to a wide range of related phenomena, including inflammation and metastasis formation. The ultimate goal of this grant proposal is to understanding the way in which platelets interact with the blood vessel wall and with other platelets via the glycoprotein receptors on their surface, and to use that knowledge to improve human health. Despite advances in analyzing the structure and function of the IIb3 receptor, which is crucial for normal hemostasis and a validated target of antithrombotic therapy, major gaps remain in understanding ligand binding and its impact on platelet physiology. Moreover, there is a need for improved potent antiplatelet therapies that cn be administered in the pre-hospital phase of myocardial infarction. The data obtained from these studies will inform attempts to develop novel inhibitors of IIb3 that have therapeutic advantages over existing agents. In Specific Aim 1 we propose to improve our understanding of ligand binding by: a) Using functional ligand binding data and new crystal structures of IIb3 as inputs to state-of-the art computational methods to identify interactions between the fibrinogen ?-module and IIb3 in addition to those made by the fibrinogen ?- chain C-terminal dodecapeptide (?C-12), b) Using electron cryomicroscopy (cryo-EM) and negative stain EM in conjunction with random conical tilt reconstructions, molecular dynamics (MD)-based flexible fitting and steered MD to obtain atomic resolution 3-dimensional (3D) representations of intact IIb3 in a nanodisc lipid bilayer in the absence of detergent. The inactive receptor, the receptor activated by talin head-domain (THD) in the absence of ligand, and THD-activated receptor in the presence of fibrinogen will each be studied. c) Using zinc finger nuclease-mediated gene editing to evaluate mutations of 3 in murine platelets rather than in cell lines that lack the platelet's signaling machinery, focusing initially on a mutation we hypothesize will enhance the binding of filamin to the 3 cytoplasmic tail and thus diminish platelet sensitivity to activation. d) Using enhanced MD techniques to characterize IIb3 activation pathways and testing the hypothesis that the new IIb3 antagonists identified in the past grant period (RUC-1, RUC-2, MSSM-1, MSSM-2) stabilize a closed, inactive conformation, thus accounting for their reduced ability to activate IIb3 compared to IIb3 antagonists patterned on the Arg-Gly-Asp (RGD) cell recognition sequence. In Specific Aim 2 we propose to identify new compounds that will provide insights into IIb3 structure-function and may have therapeutic potential by characterizing the 57 compounds (out of 126,000 tested) that most potently inhibit IIb3-mediated platelet adhesion and aggregation. We will also perform a new screen to selectively identify compounds that target ancillary fibrinogen binding sites since these may lead to new therapeutics that are safer and more efficacious.